Determination of glycated proteins

ABSTRACT

The amount of glycated proteins in a sample can be quantified by reacting the sample with first a reagent which is a combination of a protease and a peroxidase and second with a ketoamine oxidase. A kit which contains the combined peroxidase/protease enzyme reagent and also the ketoamine oxidase is also disclosed.

This invention relates to the determination of glycated proteins; moreparticularly, it relates to such a method involving the use of aproteinase in the same reagent as a peroxidase and to a kit therefor.

Horseradish peroxidase is an oxidoreductase (donor:hydrogen peroxideoxidoreductase; EC 1.11.1.7). It is widely used in the life sciences asan indicator enzyme (see, for example, Essays in Biochemistry, 1994; 28:129-146), and is one of a family of peroxidase enzymes. The particularlyuseful features of this enzyme are its ease of coupling to carriers,such as antibodies or other enzymes, its high rate of activity with arange of substrates and good thermal stability. It consists of a singlepolypeptide comprising 308 amino acids and has a relative molecular massof 44,000, which incorporates a haemin prosthetic group giving it abrown colouration. The enzyme has four disulphide bridges and containstwo calcium ions, removal of which leads to a reduction in stability.

This enzyme catalyses the transfer of hydrogen from a hydrogen donor toa hydrogen acceptor. The hydrogen acceptor is usually hydrogen peroxide,although methyl and ethyl peroxides may also be used. Hydrogen peroxideis reduced according to the following reaction:

    H.sub.2 O.sub.2 +AH.sub.2 →2H.sub.2 O+A

A wide range of hydrogen donors may be used. These include phenols,aminophenols, indophenols, diamines and leuco dyes. The oxidativeprocess of hydrogen removal from such compounds generates products whichmay be detected visually or quantified, usually in a spectrophotometer.Other means of detection used have included fluorimetry, luminometry andelectrochemistry.

The enzyme may be physically coupled to other proteins, such asantibodies or fragments thereof. This allows the specific bindingproperties of the antibody to be used to measure an analyte or toidentify histologically the location of an antigen. It may also bechemically linked to an oxidase enzyme to quantify the substrate of theoxidase. There are many analytes that may be measured using specificoxidases. Of these, several are present in biological fluids whereanalysis thereof may be clinically helpful. The use of phenol andaminoantipyrene as chromogens linked to an oxidase--peroxidase systemhas long been known (see, for example, Ann Clin Biochem, 1969, 6:24-27). More recently, alternatives to phenol, such asN-ethyl-N-(2-hydroxy-3-sulphopropyl)-m-toluidine (TOOS), have beenproposed which are more sensitive and are coloured over a wide pH range(see, for example, Chem Pharm Bull., 1982; 30: 2492-2497).

One such analyte is glycated protein, or fructosamine. This is theproduct of a non-enzymatic reaction by which glucose or other sugars mayform condensation products with free amine groups of protein (see, forexample, Clin Chem, 1987; 33: 2153-2163. In the blood, the main proteinsthat are glycated are albumin, in which exposed lysine residues providethe free amine group, and haemoglobin, in which the N-terminal valineamino acid may also react with glucose. In diabetic subjects, theconcentrations of the protein components of blood vary between fairlynarrow limits. In contrast, the glucose concentration may changesignificantly in a short period of time. Many of the pathologicalchanges experienced by diabetic patients are the consequence ofprolonged exposure of proteins to elevated glucose concentrations.Therefore, the measurement of glycated protein is clinically useful inassessing the average glucose exposure over the lifetime of the protein.

Several methods have been used to measure total glycated protein. Thecurrent reference method is the furosine procedure (see, for example, JClin Chem Clin Biochem, 1981; 19: 81-87). This involves proteindigestion in 6 molar hydrochloric acid at 95-100° C. for 18 hours.Furosine is a product of glycated lysine under these conditions and maybe measured by HPLC. This method is too complex and time-consuming forroutine use. The thiobarbituric acid procedure is slightly simpler as ituses a shorter acid digestion (2-5 hours) yielding5-hydroxymethylfurfuraldehyde, which may be reacted with thiobarbituricacid to give a derivative having an absorbance maximum at 443 nm.Another method is phenylboronate affinity chromatography. Under alkalineconditions, phenylboronate complexes with the cis-diol groups of sugars.However, even with close temperature control and prior removal ofglucose, the precision of this method is poor.

The simplest commercially available and most widely used method fordetermining serum glycated protein is based on the ability offructosamines in alkaline solution to reduce nitroblue tetrazolium (NBT)to produce a blue dye (see, for example, Clin Chem Acta, 1982; 127:87-95). The great advantage of this procedure is its ease of automation.It has since been reformulated to reduce interference due to proteinconcentration, lipids and uric acid (see, for example, Clin Chem, 1991;37: 552-556). However, only about half of the measured reducing activityin normal or well-controlled diabetics is due to glycated protein (see,for example, Clin Chem, 1988; 34: 320-323).

To avoid problems of poor specificity seen with the NBT method, anenzymatic method has been developed (see, for example EP-A-526 150).This two reagent system uses a proteinase to degrade serum protein,followed by the use of a ketoamine oxidase which acts on the glycatedfragments. The oxidase may be linked to a peroxidase and chromogensystem in an endpoint determination in which the amount of colour formedis proportional to the quantity of glycated protein in the sample. Asimilar process using an enzyme from a different source has also beendescribed (see, for example, EP-A-576 838). However, these methods arelikely to suffer significant interference from ascorbate and bilirubinwhen used with fresh samples. They also use peroxidase in a secondreagent and require a reagent blank correction.

There may also be mentioned EP-A-678576, which relates to a fructosylamino acid oxidase produced by culturing a strain of Fusarium orGibberella.

The proteinase in the first reagent must show high activity towards theglycated protein and the ability to release the substrate for theketoamine oxidase. The ability to cleave different peplide bonds isadvantageous for the rapid release of the substrate. Several proteinaseswhich are non-specific, such as pronase and proteinase K, are known,together with many other classes of proteinase having differentspecificities from a wide variety of species, which may be used alone orin combination.

Because of the requirement for an extensive proteolysis of bloodproteins, and the non-specific nature of the proteinase, the retentionof an enzyme activity in the same solution as the proteinase is mostunlikely. Therefore, the peroxidase would have to be included in thesecond of the two reagents, so that the only exposure of peroxidase tothe proteinase would be for a relatively short time in the cuvette afterthe second reagent addition. The absorbance of the cuvette is measuredjust before the addition of the second reagent and again after theketoamine oxidase/peroxidase system has produced the colour. The changein the absorbance between these two readings from a sample is due notonly to the amount of glycated protein present in the sample, but alsoto the absorbance of the second reagent itself. Therefore a blank samplemust be analysed so that a correction may be made.

For widespread acceptance of a clinical method, it is important thatinterference from substrates other than the desired analyte beminimised. Two compounds known to interfere with oxidase-peroxidasesystems are bilirubin and ascorbate (see, for example, Ann Clin Biochem,1984; 21: 398-404). The concentration of glycated protein in normalserum is about 0.1 mmol/L. The interference is more serious withanalytes present at relatively low concentration rather than analytes,such as glucose or cholesterol, with normal concentrations in excess of3 mmol/L. A high oral dose of vitamin C may cause serious interferenceeven with a cholesterol assay (see, for example, Clin Chem, 1992; 38:2160).

Different approaches have been used to reduce bilirubin interference.These include reduction of the reaction pH to 6.1 (see, for example,Clin Chem, 1981; 27: 375-379). This is not suitable for an enzymaticglycated protein method as the necessary enzymes require a higher pH.Alternatively, samples may be pretreated with peroxidase and hydrogenperoxide, which oxidises bilirubin (see, for example, Clin Chem, 1992;38: 2411-2413). However, this is not suitable as it involves an extrareagent and would be likely to interfere with the subsequent oxidasereaction. Bilirubin oxidase has also been used to remove bilirubin (see,for example, Clin Chem, 1984; 30: 1389-1392), but an extra reagent wouldbe required as the bilirubin oxidase is unlikely to retain its activityin the presence of the proteinase in the first reagent of the glycatedprotein assay.

Potassium ferrocyanide has been used to remove bilirubin interference upto 170 μmol/L in an assay for uric acid (see, for example, Clin Chem,1980; 26: 227-231). Others have shown greater removal of interference,but the incorporation of potassium ferrocyanide into the first reagentof a two reagent system caused poor reagent stability (see, for example,Clin Chem, 1993; 31: 861-868). As will be described below, it has nowbeen found that bilirubin interference up to 400 μmol/L may be removedby potassium ferrocyanide added to the first reagent of a two reagentassay for glycated protein and that the liquid reagent is stable forseveral weeks at 4° C.

Several methods have been used to protect oxidase/peroxidase systemsfrom interference by ascorbate. The most commonly used means isascorbate oxidase (see, for example, Clin Chem, 1980; 26: 227-231),which is not suitable for inclusion in a reagent containing proteinaseas it is rapidly broken down. Prior treatment with activated charcoal(see, for example, Clin Chem, 1989; 35: 2330-2333) is inconvenient.

Removal of ascorbate interference by metals having a redox potentialequal to or above that of ascorbate, but below the redox potential of achromogenic substance has been described. Such metals, including copper,might be found in Groups VIII, I-B, II-B and IV-A of the Periodic Table(see, for example, U.S. Pat. No. 3,411,887). It is important that theredox potential of the metal ion is below that of the chromogenicsubstance, otherwise the metal ion itself would generate colour in theabsence of the intended analyte. Another report tested copper in asimilar oxidase/peroxidase system, but found that only small effectswere seen even with copper concentrations as high as 30 mmol/L (see, forexample, Clin Chem, 1982; 28: 578-588).

Surprisingly, in accordance with the present invention as will bedescribed below it is possible to remove ascorbate interference in anoxidase/peroxidase method for measuring glycated protein using copper atconcentrations below 0.1 mmol/L. Furthermore, in this system, if wateris used as a sample rather than serum or plasma, copper is capable ofdirectly oxidising the chromogen system. Therefore, the redox potentialof the copper must be higher than that of the chromogen system. Thereason why copper does not interfere in the analysis of serum or plasmasamples may be due to the binding of copper by the products ofproteinase digestion of the blood proteins.

An object of the present invention is to provide an enzymatic method forthe determination of glycated protein in biological materials in whichthe peroxidase is formulated in the same reagent as the proteinase.Surprisingly, peroxidase activity is not affected by the proteinase.Other components of the second reagent do not contribute significantlyto the cuvette absorbance, so corrections using a blank sample are notrequired. The absorbance change may simply be compared to that seen witha calibrant containing a defined amount of glycated protein.

A further object of the present invention is to protect the measurementof glycated protein from interference due to ascorbate or bilirubinwhich may be present in the sample. The satisfactory removal ofascorbate interference may also depend on the inclusion of peroxidase inthe first rather than the second reagent.

The present invention may also be generally applicable to a variety ofprocesses requiring a peroxidase where it would be advantageous to mixit with a proteinase. Other applications would include cases where ananalyte has to be removed from protein to allow its measurement by anoxidase, or where intact proteins interfere with a method. The presentmethod would also be suitable for determining specific glycatedcomponents in biological fluids and for measuring glycated haemoglobin.

The present invention provides a method for the determination of aglycated protein in a sample characterised in that it comprises: mixingthe sample and a first reagent containing a proteinase and a peroxidaseso as to produce a substrate capable of oxidation by a ketoamineoxidase; adding a second reagent containing a ketoamine oxidase; andmeasuring hydrogen peroxide produced or oxygen consumed so as to detectand/or quantify the glycated protein.

The present invention also provides a kit for the determination of aglycated protein characterised in that it comprises: a first reagentcontaining a proteinase and a peroxidase; a second reagent containing aketoamine oxidase; and, optionally, means for measuring hydrogenperoxide produced or oxygen consumed.

Typically, the present methodology is applied to biological samplescomprising a body fluid, such as blood serum or plasma.

In accordance with the present invention, the proteinase is generallyproteinase K, preferably from Tritirachium album, and the peroxidase ishorseradish peroxidase. Preferably, the ketoamine oxidase is obtainablefrom the bacterial group Klebsiella, from the fungal genera Fusarium orAcremonium or from the yeast genus Debaryomyces, preferably fromFusarium. (A ketoamine oxidase catalyses the oxidation of the carbonatom in position 1 of a sugar moiety of a glycated protein withconsequent hydrolytic disruption of an amine bond to release a sugarosone and hydrogen peroxide from an amino acid.)

Commonly, the required measurement involves the use of an optionallymodified Trinder reaction (sometimes termed a "PAP" method) or an oxygenelectrode.

In preferred embodiments of the present invention, ascorbateinterference is countered by the inclusion in the first reagent of acopper (II) compound, preferably copper (II) acetate, and optionallycholic acid and/or bathophenanthroline disulphonic acid; and/orbilirubin interference is countered by the inclusion in the first and/orthe second reagent of a ferrocyanide salt, preferably potassiumferrocyanide. Furthermore, there may be included in the second reagentethylene diamine tetraacetic acid and/or mannitol with a view tomaintaining ketoamine oxidase activity.

A presently-preferred embodiment of the present invention usesproteinase K from Tritirachium album at a concentration of from 1 to 10g/L in the cuvette, together with horseradish peroxidase at a cuvetteconcentration of from 0.01 to 1 g/L.

The present invention will be further illustrated by the followingExamples:

EXAMPLE 1

Two pairs of reagents were prepared for the measurement of glycatedprotein. One pair contained peroxidase in the first reagent, togetherwith the proteinase, and the other pair contained peroxidase in thesecond reagent. The first pair contained 12 g/L proteinase K, 0.4 g/Lhorseradish peroxidase and 3.0 mmol/L 4-aminoantipyrene in 100 mmol/L of(N-2-hydroxyethyl)piperazine-N'-(3-propanesulphonic acid) (EPPS) bufferpH 8.5 in the first reagent, and 10000 U/L ketoamine oxidase and 26.6mmol/L TOOS in 100 mmol/L EPPS buffer, pH 8.5, in the second reagent.The second pair contained 12 g/L proteinase K and 3.0 mmol/L4-aminoantipyrene in 100 mmol/L EPPS buffer, pH 8.5, in the firstreagent, and 10000 U/L, ketoamine oxidase, 1.33 g/L horseradishperoxidase and 26.6 mmol/L TOOS in 100 mmol/L EPPS buffer, pH 8.5, inthe second reagent.

The reagents were tested using a Cobas Mira S autoanalyser. 100 μL ofthe reagent containing the proteinase was mixed in a plastic cuvettewith 10 μ/L of diabetic human serum and 40 μL of water diluent to washthe inside of the sample probe. After a 7 minute incubation at 37° C.,30 μL of the second reagent and 20 μL of water diluent were mixed intothe same cuvette. The cuvette absorbance was measured at 550 nm at 25second intervals from the start of the procedure until 1.5 minutes afterthe addition of the second reagent. The analyser automatically correctsthe absorbance results to account for the dilution of the cuvettecontents as the second reagent is added.

The results from the formulation in which peroxidase was protected fromthe proteinase by its addition in the second reagent are illustrated inaccompanying FIG. 1. To calculate the absorbance change that is due toglycated protein in the serum sample, the absorbance change of the watersample due to the colour of the added peroxidase must first besubtracted.

Accompanying FIG. 2 illustrates the results from the formulation inwhich the peroxidase was added as part of the first reagent. The watersample shows no increase in absorbance on the addition of the secondreagent. Despite the presence of the proteinase with the peroxidase, theabsorbance change seen with the serum sample is the same as the waterblank subtracted data of accompanying FIG. 1.

EXAMPLE 2

A reagent was prepared containing 0.2 g/L horseradish peroxidase and2.25 mmol/L 4-aminoantipyrene in 100 mmol/L EPPS buffer, pH 8.5, withand without 12 g/L proteinase K. The reagents were stored at roomtemperature. Peroxidase activity was measured in each reagent 0.5 and 24hours after the reagent was prepared by its ability to producepurpurogallin from pyrogallol and hydrogen peroxide. The results areshown below.

    ______________________________________                                        Time        Reagent with                                                                            Reagent without                                           (hours) Proteinase K Proteinase K                                           ______________________________________                                        0.5         43.9 KU/L 48.9 KU/L                                                 24 47.1 KU/L 45.3 KU/L                                                      ______________________________________                                    

Proteinase K did not reduce the activity of horseradish peroxidase inthe same solution over 24 hours at room temperature.

EXAMPLE 3

A twin reagent for the measurement of glycated protein was prepared andstored at 4° C. The first reagent contained 12 g/L proteinase K, 0.4 g/Lperoxidase, 8 mmol/L TOOS, 100 μmol/L potassium ferrocyanide, 300 μmol/Lcopper acetate and 1.2 mmol/L sodium tartrate in 75 mmol/L EPPS buffer,pH 8.0. The second reagent contained 10000 U/L ketoamine oxidase and 10mmol/L 4-aminoantipyrene in 83 mmol/L EPPS buffer, pH 8.0.

The reagents were tested with two serum samples using the Cobas Mira asin Example 1 before and after 22 days storage of the liquid reagents at4° C. The samples were stored frozen in aliquots and a freshly thawedaliquot was used for each analysis. The absorbance changes due toglycated protein in the samples were calculated by subtracting theabsorbance just before the addition of the second reagent from theabsorbance recorded 2.1 minutes later. The results are shown below.

    ______________________________________                                        Days at        Absorbance change                                              4° C.   Serum 1 Serum 2                                                ______________________________________                                         0             0.0282  0.1106                                                   22 0.0310 0.1173                                                            ______________________________________                                    

Therefore, when the complete oxidase/peroxidase assay is performed,there is no decline in the signal due to the degradation of horseradishperoxidase by proteinase K after storage of the reagent at 4° C. for 22days.

EXAMPLE 4

Three formulations for the measurement of glycated protein were preparedto demonstrate the effect of peroxidase on the ability to removeascorbate and bilirubin interference. In formulation A, the firstreagent contained 12 g/L proteinase K, 0.4 g/L peroxidase and 8 mmol/LTOOS in 75 mmol/L EPPS buffer, pH 8.0. The second reagent contained10000 U/L ketoamine oxidase and 10 mmol/L 4-aminoantipyrine in 83 mmol/LEPPS buffer, pH 8.0. This formulation contained no ingredients to combatthe interferences. Formulation B was the same as A, with the addition of100 μmol/L potassium ferrocyanide, 300 μmol/L copper acetate and 1.2mmol/L sodium tartrate to the first reagent. These additions were toreduce interference from bilirubin and ascorbate. Formulation C was thesame as B, except that there was no peroxidase in the first reagent. Thesecond reagent contained peroxidase reagent at a concentration of 1.33g/L. Therefore, after the mixing of sample, reagents and diluents on theanalyser according to the protocol of Example 1, all three formulationshad the same cuvette concentration of peroxidase.

The three formulations were used to assay for glycated protein in foursamples. These were: (1) water, (2) a control serum dilution (one partof water to four parts of serum), (3) serum diluted in the same way witha stock solution of ascorbate such that the concentration of ascorbatein the serum was 400 μmol/L, (4) serum diluted with a stock solution ofunconjugated bilirubin such that the concentration of bilirubin in theserum was 400 μmol/L. In the calculation of results with formulation C,the absorbance change seen with the water sample was subtracted from theabsorbance changes given by the serum samples to correct for theabsorbance due to the peroxidase in the second reagent.

In the Table below, the effects of ascorbate and bilirubin interferenceare shown as the absorbance change seen with that interference expressedas a percentage of the absorbance change given by the control serum.

    ______________________________________                                                     Percentage recovery in serum with:                                              400 μmol/L                                                                           400 μmol/L                                          Formulation ascorbate bilirubin                                             ______________________________________                                        A               9        72                                                     B 95 97                                                                       C 540  99                                                                   ______________________________________                                    

Bilirubin at a concentration of 400 μmol/L reduced the absorbance changeto 72% of the control sample when used with Formulation A. However, thecombination of ferrocyanide, copper and tartrate in the first reagentalmost abolished this interference in Formulation B (with peroxidase inthe first reagent) and Formulation C (with peroxidase in the secondreagent).

Ascorbate interference was particularly severe in Formulation A, withthe loss of over 90% of the signal. The additional ingredients ofFormulation B reduced this effect to less than 5%. However, when thesame amount of peroxidase was added in the second reagent (FormulationC), there was a massive increase in the absorbance change. Therefore, inthis system the removal of ascorbate interference depends on theaddition of the peroxidase in the first reagent.

EXAMPLE 5

In order to maximise laboratory efficiency, it is desirable that testsshould take as little time as possible on an autoanalyser. Indeed, someanalysers are incapable of running two reagent chemistries with a firstincubation time of greater than three minutes. The detrimental effect ofreducing the incubation time of the sample with the first reagent on theability to remove ascorbate interference is illustrated below withformulation A. Formulation B, however, contains some additionalcomponents which significantly improve interference removal.

In formulation A, the first reagent contained 6 g/L proteinase K, 0.4g/L peroxidase, 8 mmol/L TOOS, 20 μmol/L potassium ferrocyanide, 250μmol/L copper acetate and 1.0 mmol/L sodium tartrate in 75 mmol/L EPPSbuffer, pH 8.0. The second reagent contained 10000 U/L ketoamine oxidaseand 10 mmol/L 4-aminoantipyrene in 83 mmol/L EPPS buffer, pH 8.0. Thisformulation was tested using two different incubation times, 2.9 and 7minutes.

In formulation B, the first reagent contained 6 g/L proteinase K, 0.4g/L peroxidase, 8 mmol/L TOOS, 100 μmol/L potassium ferrocyanide, 100μmol/L copper acetate, 2% w/v cholic acid, 1% w/v polyoxyethylene 10tridecyl ether and 175 μmol/L bathophenanthroline disulphonic acid in 75mmol/L EPPS buffer, pH 8.0. The second reagent was the same as informulation A.

The two formulations were used to assay for glycated protein in threesamples, viz: (1) a control serum dilution (by volume, 1 part of waterto 4 parts of serum); (2) serum diluted in the same way with a stocksolution of ascorbate so that the concentration of ascorbate in theserum was 300 μmol/L; (3) serum diluted with a stock solution ofunconjugated bilirubin so that the concentration of bilirubin in theserum was 300 μmol/L.

Sample, reagents and diluents were mixed on the analyser according tothe protocol of Example 1, except that the incubation of sample andfirst reagent was for either 2.9 or 7 minutes.

In the Table below, the effects of ascorbate and bilirubin interferentsare shown as the absorbance change seen with that interferent expressedas a percentage of the absorbance change given by the control serum.

    ______________________________________                                               2.9 minute        7 minute                                               incubation % recovery  incubation % recovery                                  in serum with:  in serum with:                                                       400      400        400    400                                         Formu- μmol/L μmol/L μmol/L μmol/L                                lation ascorbate bilirubin ascorbate bilirubin                              ______________________________________                                        A        82       87         90     97                                          B 95 95                                                                     ______________________________________                                    

EXAMPLE 6

A disadvantage of the shorter incubation time of sample and reagent 1,using reagents which overcome the interferences referred to above, isthat reaction rates are seen when water is used as the sample. Althoughthis background rate is not apparent when serum is the sample, it ispreferable that the method should work with samples other than serum.

In formulation A, the first reagent contained 4 g/L proteinase K, 0.28g/L peroxidase, 5.6 mmol/L TOOS, 90 μmol/L potassium ferrocyanide, 90μmol/L copper acetate, 1.8% w/v cholic acid, 1.2% w/v polyoxyethylene 10tridecyl ether and 144 μmol/L bathophenanthroline disulphonic acid and 5mmol/L calcium acetate in 75 mmol/L EPPS buffer, pH 8.0. The secondreagent contained 13000 U/L ketoamine oxidase and 10.5 mmol/L4-aminoantipyrene in 50 mmol/L EPPS buffer, pH 8.0.

Formulation B was the same as formulation A, except that 30 mmol/Ldisodium EDTA was included in the second reagent.

The two formulations were used to assay for glycated protein in threesamples, viz: (1) water; (2) serum from a diabetic subject; (3) plasmafrom a diabetic subject.

The formulations were tested on a Cobas Mira S autoanalyser. The reagentcontaining the proteinase (250 μL) was mixed in a plastic cuvette with20 μL of sample and 30 μL of water diluent to wash out the inside of thesample probe. After a 2.9 minute incubation at 37° C., 50 μL of thesecond reagent and 10 μL of water diluent were mixed into the samecuvette. The cuvette absorbance was measured at 550 nm at 25 secondintervals from the start of the procedure until 1.5 minutes after theaddition of the second reagent.

The absorbance profiles seen with formulations A and B are illustratedin accompanying FIGS. 3 and 4, respectively. It may be seen that thepresence of EDTA in the second reagent prevents the increase inabsorbance seen after the addition of the second reagent when water isthe sample. The signal given by the serum and plasma sample is 15%greater when EDTA is included in the second reagent. Both these effectsmay be due to the chelation of copper by EDTA. Copper is able to producea signal with the chromogens and also partially inhibits ketoamineoxidase activity.

EXAMPLE 7

The second reagent may be stabilised by the inclusion of mannitol.

Two formulations of the second reagent were prepared. Formulation Acontained 50 mmol/L EPPS buffer, pH 8.0; 10.5 mmol/L aminoantipyrene; 30mmol/L EDTA and 6000 U/L ketoamine oxidase. Formulation B was the sameapart from the inclusion of 5% mannitol. After lyophilisation andreconstitution with demineralised water, each formulation was stored for21 days both at 25° C. and frozen at -20° C. as a control.

The stability of the reagent formulations with and without mannitol wastested using the Cobas Mira protocol of Example 6 with serum as thesample and a first reagent composed of 3 g/L proteinase K, 0.28 g/Lperoxidase, 5.6 mmol/L TOOS, 90 μmol/L potassium ferrocyanide, 90 μmol/Lcopper acetate, 1.8% cholic acid, 1.2% polyoxyethylene 10 tridecylether, 144 μmol/L bathophenanthroline disulphonic acid and 5 mmol/Lcalcium acetate in 60 mmol/L EPPS buffer, pH 8.0. Results werecalculated by subtracting the absorbance of the cuvette at 550 nm justbefore the addition of the second reagent from the absorbance measured2.5 minutes after the second reagent addition.

The absorbance changes after 21 days were as follows:

    ______________________________________                                        Storage     Formulation A     Formulation B                                   temperature Abs change                                                                              %       Abs change                                                                            %                                       ______________________________________                                        -20° C.                                                                            0.1036            0.1062                                            +25° C. 0.0795 77 0.1040 98                                          ______________________________________                                    

Over three weeks at 25° C., the signal produced using Formulation A hadfallen to only 77% of the control frozen reagent. However, FormulationB, which contained mannitol, was stable.

EXAMPLE 8

The application of the enzymatic glycated protein method was testedalongside the commercially available nitroblue tetrazolium method (Rochecatalogue number 0736694) by comparison with a furosine referenceprocedure. This involved acid hydrolysis, followed by HPLCquantification of furosine, specific for protein glycation. Fructosyllysine was used as a standard.

The formulation of the enzymatic reagent was as follows:

The first reagent contained 4 g/L proteinase K, 0.28 g/L peroxidase, 5.6mmol/L TOOS, 90 μmol/L potassium ferrocyanide, 30 μmol/L copper acetate,1.8% cholic acid, 0.25% polyoxyethylene-10-tridecyl ether, 144 μmol/Lbathophenanthroline disulphonic acid and 5 mmol/L calcium acetate in 60mmol/L EPPS buffer, pH 8.0. The second reagent contained 10.5 mmol/Laminoantipyrene, 30 mmol/L EDTA, 9000 U/L ketoamine oxidase and 3%mannitol in 50 mmol/L EPPS buffer, pH 8.0.

250 μL of the first reagent was mixed in a plastic cuvette with 20 μL ofsample and 30 μL of water diluent to washout the inside of the sampleprobe. After a 5 minute incubation at 37° C., 50 μL of the secondreagent and 10 μL of water diluent were mixed into the same cuvette. Thecuvette absorbance was measured at 550 nm at 25 second intervals for atotal of 10 minutes. The absorbance change due to glycated protein inthe sample was calculated by subtracting the absorbance just before theaddition of the second reagent from that measured 5 minutes afterwards.

56 serum samples from diabetic subjects were assayed for glycatedprotein by each of the three methods. Both enzymatic and NBT methodscorrelated well with the reference method, r=0.95 and 0.96,respectively, (see accompanying FIG. 5). The NBT method showed apositive bias of 95 μmol/L, or 34% of the quoted upper reference limit,while the enzymatic regression line passed very close to the origin.This suggests that both the enzymatic and NBT methods are measuring thesame analyte, but that the enzymatic procedure is not subject to anon-specific background reducing activity present in serum.

What is claim is:
 1. A method for the determination of a glycatedprotein in a sample comprising the steps of: a) mixing the sample with afirst reagent containing a proteinase and a peroxidase; b) incubatingthe mixture of step a) for a time sufficient to produce a substratecapable of oxidation by a ketoamine oxidase; c) adding a second reagentcontaining a ketoamine oxidase to the incubated mixture of step b); andd) measuring hydrogen peroxidase produced or oxygen consumed in step c)to quantify the glycated protein present in the sample.
 2. A method asclaimed in claim 1 wherein the sample is blood serum or plasma.
 3. Amethod as claimed in claim 1 or claim 2 wherein the proteinase isproteinase K.
 4. A method as claimed in any of claim 1 wherein theperoxidase is horseradish peroxidase.
 5. A method as claimed in any ofclaim 1 wherein the ketoamine oxidase is obtainable from the bacterialgroup Klebsiella, from the fungal genera Fusarium or Acremonium or fromthe yeast genus Debaryomyces.
 6. A method as claimed in any of claim 1wherein the measurement involves the use of an optionally modifiedTrinder reaction or an oxygen electrode.
 7. A method as claimed in anyclaim 1 wherein ascorbate interference is countered by the inclusion inthe first reagent of a copper (II) compound, a cholic acid or abathophenanthroline disulphonic acid or a mixture thereof.
 8. A methodas claimed in any of claim 1 wherein bilirubin interference is counteredby the inclusion in the first and/or second reagent of a ferrocyanidesalt.
 9. A method as claimed in any of claim 1 wherein the secondreagent includes ethylene diamine tetraacetic acid and/or mannitol. 10.A kit for the determination of glycated protein comprising: a firstreagent containing a proteinase and a peroxidase; a second reagentcontaining a ketoamine oxidase.